The packaging of the genome into chromatin is essential for normal growth, development, and differentiation. Chromatin is a dynamic structure that tightly regulates all of the processes that use DMA as a substrate, including transcription, DMA replication, DMA repair, and recombination. Furthermore, chromatin assembly and disassembly processes are important in human disease, as seen in the multiple genetic malformations, as well as cancer and leukemia subtypes that have been linked to aberrations in proteins that modify chromatin structure. The key proteins responsible for chromatin disassembly and chromatin assembly are the histone H3 and H4 chaperones Anti-silencing function 1 (Asf1) and the Chromatin Assembly Factor (CAF-1) complex. These proteins are highly conserved throughout eukaryotic evolution and are the focus of this study because of their central role in histone H3/H4 deposition and nucleosome disassembly activities. The long-term goal of this project is to gain a unified understanding of the relationship among histone H3/H4 chaperones and their mechanism of chromatin assembly and disassembly in vitro and in vivo. The first Aim is to define the determinants of Asfl -mediated histone H3/H4 chaperone function using molecular genetics and structural approaches. The second Aim is to investigate the molecular mechanism of the H3/H4 chaperone activity of Asfl using biophysical analyses. The third Aim is to gain key insight into the function of the central DNA replication-dependent chromatin assembly factor - the trisubunit CAF-1 complex, and its interactions with Asfl to establish the molecular mechanism of chromatin assembly. These studies will take advantage of our recent Asf1-H3/H4 crystal structure, recombinant chromatin assembly factors that we have already generated, and physiological analyses in the budding yeast model system. By combining and applying the individual expertise of the PI and Co-Pi in biophysics, structural biology, genetics and biochemistry to this collaborative study of histone H3/H4 chaperones, we are in a unique position to make important strides toward a detailed understanding of the molecular basis of histone chaperone activity. This work will fill critical gaps in the current understanding of these fundamental processes of chromatin assembly and disassembly, which are essential and central to all DMA-dependent cellular functions. Relevance to the public health. Many diseases are the result of incorrect gene expression. The molecular and structural understanding of chromatin assembly and disassembly that will come from this work will further our ability to modify the epigenetic codes involved in human diseases for the purpose of therapeutic intervention.